1. Field of the Invention
The invention relates to the field of modified antibodies, derivatives or fragments thereof.
2. Description of Related Art
The pharmaceutical industry increasingly encounters the need for cost effective alternative large scale production systems of biopharmaceuticals. Plant-based expression systems have meanwhile demonstrated their usefulness as a suitable alternative to animal cell factories. Especially, their low production costs combined with exceptional safety through minimized risks of contamination due to the absence of human pathogens (Raskin, I. et al., Trends Biotechnol 20, 522-531 (2002); Fischer, R. et al., Curr Opin Plant Biol 7, 152-158 (2004)) is of utmost importance. Plants are able to perform most of the higher eukaryotic posttranslational modifications (Gomord, V. & Faye, L., Curr Opin Plant Biol 7, 171-181 (2004)). These include complex glycosylations, protein processing and folding as well as the assembly of complex multimeric proteins, features that contribute to the bioactivity and the pharmacokinetics of active therapeutic antibodies. Hence, various recombinant proteins, including human antibodies, have been expressed successfully in plant host expression systems (Hiatt, A. et al., Nature 342, 76-78 (1989); Ma, J. K. et al., Nat Rev Genet 4, 794-805 (2003)).
Nevertheless plant derived N-linked oligosaccharides differ considerably from those found in humans. Besides the general absence of α1,6-fucosyl residues in plants, differences in post-translational modifications, such as glycosylation, have been shown to influence the properties of plant-derived proteins (Daniell et al., supra; Conrad et al. (1998) Plant Mol. Biol. 38:101-109; Mann et al. (2003) Nat. Biotechnol. 21:255-261). In plants, N-linked glycans may contain antigenicS (Faye et al. (1993) Anal. Biochem. 109:104-108) and/or allergenic (van Ree et al. (2000) J. Biol. Chem. 275:11451-11458) β(1,2)-xylose (Xyl) residues attached to the N-linked Mannose of the glycan core and α(1,3)-fucose (Fuc) residues linked to the proximal GlcNAc that are not present on mammalian glycans. In contrast sialic acid residues are normally not attached to plant N-glycans. However, plant antibodies do not require these residues for successful topical passive immunization (Ma et al., supra).
Glycosylation processing in the endoplasmic reticulum (ER) is conserved amongst almost all species and restricted to oligomannose (Man5-9GlcNAc2) type N-glycans, whereas the Golgi-generated processing to hybrid and complex type glycans is highly diverse (Helenius et al. (2001) Science 291:2364-2369). ER retention of expressed proteins in transgenic plants usually improves the production levels (Conrad et al. (1998) Plant Mol. Biol. 38: 101-109; Sharp et al. (2001) Biotechnol. Bioeng. 73:338-346). However, since glycan processing can affect the stability of antibodies (Rudd et al. (2001) Science 291:2370-2376), it is unclear whether an antibody derived from plant expression systems with modified glycan structures would be active and able to confer effective systemic post-exposure prophylaxis.
As large-scale compatible production platform for recombinant proteins in contained suspension cultures the robust moss Physcomitrella patens offers an absolute animal component free, next generation production technology by combining several beneficial attributes with an—not only among land plants—extraordinarily high rate of homologous nuclear DNA recombination allowing an efficient targeted knockout of genes (Gorr, G. & Wagner, S., Modern Biopharmaceuticals 3, 919-929 (2005); Girke, T. et al., Plant J 15, 39-48 (1998); Schaefer, D. G. & Zyrd, J. P; Plant J 11, 1195-1206 (1997). In attempts to “humanize” N-linked oligosaccharide structures, double knockout variants for β1,2-xylosyltransferase and α1,3-fucosyltransferase genes (Δxyl-t/Δfuc-t) have recently been generated according to WO 04/057002. These moss variants synthesized total glycoproteins completely lacking the two plant-specific sugar residues yet they were not affected in morphology, growth, development and the ability to secrete recombinant glyco-proteins (Koprivova, A. et al.; Plant Biotechnol J 2, 517-523 (2004); Huether, C. M. et al. Plant Biol 7, 292-299 (2005)). The successful attachment of terminal, human-like 1,4 linked galactose to N-glycans from moss has been shown also (Huether, C. M. et al. Plant Biol 7, 292-299 (2005); Gorr and Jost Bioprocess J 4, 26-30 (2005)). Functional characteristics of the antibodies like ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity) activity were not disclosed there.
Although there have been attempts to produce antibodies by plant expression systems, stability of the antibodies due to changed glycosylation patterns and negative effects on effector function and interaction between Fc regions and Fc receptors of these antibodies have been described. Functions mediated by the Fc-part of immunoglobulins, have been reported to be strongly related to their N-linked oligosaccharide structures (Jefferis, R. et al., Immunol Rev 163, 59-76 (1998)).
Particularly core fucosylated oligosaccharides showed weaker binding to the FcγUIIIa receptor (CD16) expressed on effector cells and resulted in a decreased lytic potential (Shields, R. L. et al., J Biol Chem 277, 26733-26740 (2002); Shinkawa, T. et al., J Biol Chem 278, 3466-3473 (2003)). In contrast, yeast produced antibodies lacking core fucose in its N-glycan pattern showed weak potential in a B-cell depletion assay. Only high concentrations of the antibody resulted in a depletion of B-cells from a healthy donor. Characteristics of the antibodies like ADCC and CDC activity were not disclosed there.
However, following the production of the antibody in vivo most of the N-glycan structures presented in this study were processed in vitro in further steps by the use of specific enzymes to achieve the final N-glycan patterns (Li et al., Nat Biotechnol, doi: 10.1038/nbt1178 (2006).
The U.S. Pat. No. 6,602,684 describes methods to increase the effector function of an antibody by modifying complex glycan structures, such as bisected N-linked glycan structures modified by GnTIII.
Monoclonal antibodies against rabies are described in the WO 2005/000225 A2. These antibodies are of the IgG, IgA, IgM, IgD and IgE class, are produced in plants lack N-glycan structures with alpha-1,3-fucose residues and have less allergenic plant epitopes.
The WO 2004/050838 A2 describes immunoglobins against herpes simplex virus produced in plants without fucose residues but may comprise xylose.
The disclosure of the WO 01/31045 A1 relates to a method of producing proteins with mammal-like glycostructure in plants. Preferably the plants do not have an active fucosyltransferase or xylosyltransferase.
The US 2006/0034829 A1 describes immunoglobins with a N-glycan structure of the formula Man3GlcNAc2.
The US 2006/0029604 A1 describes immunoglobins with a N-glycan structure of the formula GlcNac2Man3GlcNac2. These structures are generated by β-galactosidase treatment.
The WO 01/55434 A1 relates to the inhibition of carbohydrate modifying enzymes in plants, in particular GBSS and GnTI.
Even in view of the long and intensive research on development of antibodies, there is still a high demand for antibodies with improved characteristics like increased effector functions.